Muratori Monica, Tamburrino Lara, Marchiani Sara, Cambi Marta, Olivito Biagio, Azzari Chiara, Forti Gianni, Baldi Elisabetta
Sexual Medicine and Andrology Unit, Department of Experimental and Biomedical Sciences, Center of Excellence DeNothe, University of Florence, Italy.
Pediatric Section, Department of Health Sciences, University of Florence and Anna Meyer Children's University Hospital, Florence, Italy.
Mol Med. 2015 Jan 30;21(1):109-22. doi: 10.2119/molmed.2014.00158.
Sperm DNA fragmentation (sDF) represents a threat to male fertility, human reproduction and the health of the offspring. The causes of sDF are still unclear, even if apoptosis, oxidative assault and defects in chromatin maturation are hypothesized. Using multicolor flow cytometry and sperm sorting, we challenged the three hypothesized mechanisms by simultaneously evaluating sDF and signs of oxidative damage (8-hydroxy, 2'-deoxyguanosine [8-OHdG] and malondialdehyde [MDA]), apoptosis (caspase activity and cleaved poly[ADP-ribose] polymerase [cPARP]) and sperm immaturity (creatine phosphokinase [CK] and excess of residual histones). Active caspases and c-PARP were concomitant with sDF in a high percentage of spermatozoa (82.6% ± 9.1% and 53.5% ± 16.4%, respectively). Excess of residual histones was significantly higher in DNA-fragmented sperm versus sperm without DNA fragmentation (74.8% ± 17.5% and 37.3% ± 16.6%, respectively, p < 0.005), and largely concomitant with active caspases. Conversely, oxidative damage was scarcely concomitant with sDF in the total sperm population, at variance with live sperm, where 8-OHdG and MDA were clearly associated to sDF. In addition, most live cells with active caspase also showed 8-OHdG, suggesting activation of apoptotic pathways in oxidative-injured live cells. This is the first investigation on the origin of sDF directly evaluating the simultaneous presence of the signs of the hypothesized mechanisms with DNA breaks at the single cell level. The results indicate that the main pathway leading to sperm DNA breaks is a process of apoptosis, likely triggered by an impairment of chromatin maturation in the testis and by oxidative stress during the transit in the male genital tract. These findings are highly relevant for clinical studies on the effects of drugs on sDF and oxidative stress in infertile men and for the development of new therapeutic strategies.
精子DNA碎片化（sDF）对男性生育能力、人类生殖及后代健康构成威胁。尽管推测其原因与细胞凋亡、氧化损伤及染色质成熟缺陷有关，但sDF的成因仍不明确。我们运用多色流式细胞术和精子分选技术，通过同时评估sDF以及氧化损伤迹象（8-羟基-2'-脱氧鸟苷[8-OHdG]和丙二醛[MDA]）、细胞凋亡（半胱天冬酶活性和裂解的聚[ADP-核糖]聚合酶[cPARP]）和精子不成熟（肌酸磷酸激酶[CK]和残留组蛋白过量），对这三种推测机制进行了验证。在高比例的精子中，活性半胱天冬酶和c-PARP与sDF同时存在（分别为82.6%±9.1%和53.5%±16.4%）。与无DNA碎片化的精子相比，DNA碎片化精子中的残留组蛋白过量显著更高（分别为74.8%±17.5%和37.3%±16.6%，p<0.005），且与活性半胱天冬酶在很大程度上同时存在。相反，在整个精子群体中，氧化损伤与sDF几乎不同时出现，这与活精子不同，在活精子中8-OHdG和MDA与sDF明显相关。此外，大多数具有活性半胱天冬酶的活细胞也显示出8-OHdG，这表明氧化损伤的活细胞中凋亡途径被激活。这是首次在单细胞水平上直接评估推测机制的迹象与DNA断裂同时存在情况的sDF起源研究。结果表明，导致精子DNA断裂的主要途径是细胞凋亡过程，可能由睾丸中染色质成熟受损以及在男性生殖道运输过程中的氧化应激触发。这些发现对于不育男性中药物对sDF和氧化应激影响的临床研究以及新治疗策略的开发具有高度相关性。
